Acetylation modifies protein subcellular localization, stability, enzymatic activity, and protein-protein and protein-DNA interactions, playing a crucial role in mediating protein function. However, research on non-histone acetylation remains limited. This study investigates changes in lysine acetylation (Kac) in proteins in response to drought using 4D label-free quantitative lys-acetylproteome analysis. We identified a total of 15,064 acetylated peptides across 4,393 proteins, with 2,486 Kac sites exhibiting significant changes: 246 proteins showed increased Kac levels, while 1,406 displayed reductions. Notably, proteins associated with metabolic pathways, such as nucleotide sugar biosynthesis, proteasome, glutamate decarboxylase and the tricarboxylic acid (TCA) cycle, were significantly impacted. The alterations in Kac levels correlated with various KEGG pathways, suggesting that acetylation plays a regulatory role in drought response mechanisms. Furthermore, we identified specific acetylation sites in transcription factors (TFs), highlighting their involvement in this process. Functional validation demonstrated that mutations in Kac sites of five randomly selected TFs resulted in significant changes in drought tolerance, emphasizing the critical role of lysine acetylation in modulating stress responses. Overall, our findings indicate that Kac modification serves as a key regulatory mechanism in birch adaptation to drought stress, influencing both metabolic processes and transcriptional regulation.